Many methods and apparatuses have been developed for use in procedures in which substances desired to be transferred into cells are transferred into cells, particularly, gene recombination procedures in which genes such as DNA and RNA are introduced into cells.
For example, competent cells are often used for bacteria and yeasts which have cell walls containing peptide glucan, cellulose and the like (for example, refer to Non-Patent Document 1). This is a method in which cells are altered so as to easily take up substances, and then heat shock or the like is applied to the cells so as to allow the substance to be taken up. However, this method is problematic in that it is complex and time-consuming because conditions for culture of cells to be used must be strictly regulated, and cells must be frozen or treated with a solution containing metal ions without killing the cells (for example, refer to Non-Patent Documents 2 and 3).
Animal cells usually proliferate more slowly and have greatly different cell structures and cell membrane structures than bacteria and other microorganisms. Therefore, it is generally said to be difficult to transfer a substance into animal cells as compared with microorganisms, and very fine handling is required for animal cells. Therefore, transfer into animal cells of genes such as DNA and RNA or other substances to be transferred into cells cannot be made without difficulty.
Several methods targeting animal cells have been proposed. One example is a calcium phosphate method (for example, refer to Non-Patent Document 4). This is a method in which calcium phosphate particles containing DNA are made and allowed to be taken up via endocytosis. However, this method has drawbacks such that procedures are complex, and transfer efficiency is low. As a modification of this method, a method has been proposed in which substances are allowed to be taken up into cells using reagents such as cationic polymers and liposomes (for example, refer to Non-Patent Documents 5 and 6). However, this method is problematic in that very complex procedures are required to mix reagents with cells, and the reagents to be used are expensive. Furthermore, these reagents may exert a fatal action on cells depending on doses and can only be used within a limited range of concentrations.
In addition to these methods, methods in which viruses are utilized to transfer substances into cells are available (for example, refer to Non-Patent Document 7). However, complex purification procedures are required when viruses are used. Because of viruses, there is a risk that substances may be transferred into cells or tissues that are not targeted. Furthermore, because of viruses, biohazard risk must be considered.
Electroporation is a well-known method by which a substance to be transferred into cells is allowed to be taken up into cells (for example, refer to Patent Document 1). This is a method in which a high pulsed voltage is applied to a suspension containing a gene and a cell so as to allow the gene contained in the suspension to be taken up into the cell. This method is wide in application and high in gene transfer efficiency. However, since gene transfer efficiency has a proportional relationship with cell fatality rate, the transfer of substances cannot be carried out independently of cell fatality. Specifically, the method has drawbacks such that, if pulse conditions are inappropriate, not only targeted substances are hardly transferred into cells, but also cells may die. Furthermore, in order to prevent so-called towing which is electric discharge into a solution, electrical conductivity of the cell suspension must be reduced when a voltage is applied. For this purpose, procedures of isolating and washing cells with centrifugation or the like are required, during which cells may die.
Furthermore, a particle gun is used as a method for transferring a substance into cells (for example, refer to Patent Document 2). This is a method in which gene-carrying microparticles of gold, tungsten or the like are shot into cells. The substance to be transferred into cells is carried by microparticles so as to increase the whole mass, and is allowed to hit cells with high energy so as to penetrate through cell membranes. This method shows high transfer efficiency against cells hit by microparticles, but has drawbacks such that, when there are lots of cells, microparticles do not uniformly hit the cells, thereby making it difficult to increase the total transfer efficiency. Furthermore, this method also has drawbacks such that a procedure of attaching genes to microparticles or the like is required, and consumable materials such as microparticles for carrying substances and rupture discs for releasing pressure are expensive. Furthermore, there are also drawbacks such that an apparatus is bulky because a plasma explosion, an explosive or a compressed gas cylinder must be used to shoot microparticles, cells are scattered by the blast of gas, cells are easily damaged because particles remain in cells, particles are accumulated in cells after shooting is repeated, and so forth. The retention and accumulation of microparticles in cells are not desirable particularly for gene therapy because probability of cell death is increased. Furthermore, since sizes of microparticles to be used are from 0.5 to 3 μm, it is very difficult to apply this method to, for example, minute cells such as bacteria having a size of around 1 μm, although relatively large cells such as of plants and animals having a size of 10 to 100 μm have little problem.
Electrospray is used as a method for spraying a liquid at high speed. Electrospray is a method in which electric charges are collected at the tip of a spray tube by applying a high voltage to the tube, and a liquid is passed through the tube tip at which the electric charges have been accumulated, thereby allowing the liquid to be converted into minute charged droplets and sprayed on a target at high speed. This method has been used specifically as an ionization method in mass spectrometry (for example, refer to Non-Patent Documents 8 and 9).
As one of the particle gun techniques utilizing this electrospray, there is a method in which a high voltage is applied to a suspension containing particles through the tip of a capillary in order to spray them on cells (for example, refer to Patent Document 3). This method uses the electrospray in order to accelerate particles to which a substance to be transferred into cells is attached or a liquid containing the substance together with the particles. In either case, this method has a drawback such that the capillary tip may be clogged with particles because the suspension containing particles is used. Furthermore, like usual particle guns, the method has a drawback such that solids remain in cells. Furthermore, every time when the substance to be transferred into cells is changed, the procedure for carrying the substance must be done, although this is unnecessary when one type of a substance desired to be transferred into cells is to be transferred. Furthermore, the liquid transfer system for the suspension must be subjected to washing and replacement every time they are used, and problems of complex procedures and time-consumption still remain unsolved. Furthermore, since a suspension containing a substance to be transferred is sprayed, the inside of an apparatus may be contaminated. Furthermore, spark discharge is more likely to occur between the tube and cells as the amount of a solute contained in a spray solution is increased.
Under such circumstances of background art, provision of a method and an apparatus has been demanded in which a substance to be transferred into cells can be conveniently and continuously transferred into cells as it is without the need of attaching the substance to be transferred into cells to carriers such as particles, and in which multiple types of substances to be transferred into cells can be continuously transferred into multiple types of cells in a short time.    Patent Document 1: U.S. Pat. No. 4,945,050.    Patent Document 2: Japanese Patent No. 2606856.    Patent Document 3: U.S. Pat. No. 6,093,557.    Non-Patent Document 1: H. Inoue, H. Nojima, H. Okayama, Gene, 1990, vol. 96(1), p 23-28.    Non-Patent Document 2: J. Haensler, F. C. Szoka Jr, Bioconjugate Chem., 1993, vol. 4, p 374-379.    Non-Patent Document 3: P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, 1147766570484—0.′); 1987, vol. 84(21), p 7413-7.    Non-Patent Document 4: F. L. Graham, A. J. Van Der Eb, Virology, 1973, vol. 52, p 446-467.    Non-Patent Document 5: J. Haensler, F. C. Szoka Jr, Bioconjugate Chem., 1993, vol. 4, p 374-379.    Non-Patent Document 6: P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, 1147765309078—0.′); 1987, vol. 84(21), p 7413-7.    Non-Patent Document 7: D. Yu, T. Shioda, A. Kato, M. K. Hasan, Y. Sakai, Y. Nagai, 1147765309078—1.′); 1997, vol. 2(7), p 457-66.    Non-Patent Document 8: J. B. Fenn, M. Mann, C. K. Meng, S. F. Wong, C. M. Whitehouse, Science, 1989, vol. 246, p 64-71.    Non-Patent Document 9: Z. Takats, J. M. Wiseman, B. Gologan, R. G. Cooks, Science, 2004, vol. 306, p 471-473.